Anthrax is a potentially lethal disease, caused by Bacillus anthracis. It poses a significant threat as an agent of biological warfare and terrorism. Current therapy of anthrax is usually preventative, in form of a vaccine or, in case of acute infection, treatment with antibiotics. However, antibiotics alone typically fail against systemic anthrax unless administered at an early stage, since they do not neutralize the toxins produced by the bacterium. Consequently, there is a great need for small molecule toxin inhibitors. In combination with antibiotic therapy, such inhibitors could be life-saving. Of all three toxins produced by B. anthracis, we have chosen the Zinc proteinase 'lethal factor' (LF) as the target for inhibitor development, since it appears to be the critical virulence factor. In situ click chemistry, an exploratory new technique in which the target protein assembles its own inhibitors from simple precursors, will be employed for lead discovery and optimization. The main goals of the project are to (a) investigate the use of in situ click chemistry for the discovery and optimization of Anthrax LF inhibitors; (b) develop a detailed understanding of the structural requirements for LF inhibition, by structure-activity profiling and molecular modeling; (c) develop compounds that selectively inhibit Anthrax LF with high affinity (<1 mu M) and that are active in cell-based assays. Hits and leads will be optimized with the aid of target and cell-based inhibition assays, which will be accessed through collaborations. The specific aims are as follows: (1) Lead Discovery: Development of Anthrax LF inhibitors based on in situ click chemistry; (2) Lead Optimization: Optimization of binding affinity and cell-based activity.